Glaucoma is a common ocular disorder that is a leading cause of blindness worldwide. It is characterized by the dysfunction and loss of retinal ganglion cells (RGCs). Although many studies have implicated various molecules in glaucoma, no mechanism has been shown to be responsible for the earliest detectable damage to RGCs and their axons in the optic nerve. Here, we show that the leukocyte transendothelial migration pathway is activated in the optic nerve head at the earliest stages of disease in an inherited mouse model of glaucoma. This resulted in proinflammatory monocytes entering the optic nerve prior to detectable neuronal damage. A 1-time x-ray treatment prevented monocyte entry and subsequent glaucomatous damage. A single x-ray treatment of an individual eye in young mice provided that eye with long-term protection from glaucoma but had no effect on the contralateral eye. Localized radiation treatment prevented detectable neuronal damage and dysfunction in treated eyes, despite the continued presence of other glaucomatous stresses and signaling pathways. Injection of endothelin-2, a damaging mediator produced by the monocytes, into irradiated eyes, combined with the other glaucomatous stresses, restored neural damage with a topography characteristic of glaucoma. Together, these data support a model of glaucomatous damage involving monocyte entry into the optic nerve. Genome-wide assessment of gene expression changes was performed in DBA/2J-Gpnmb+, DBA/2J mice and irradiated DBA/2J mice at 8.5 and 10.5 months of age. Overall design: In this study (Howell et al, JCI, 2012), 50 samples (10 D2-Gpnmb+ control at 8.5 mos, 20 NOE DBA/2J at 8.5 mos, 10 radiation-treated DBA2J at 8.5 mos and 10 radiation-treated DBA/2J at 10.5 mos) were combined with 30 of ONH samples from the GSE26299 study (10 D2-Gpnmb+ and 20 NOE DBA/2J all at 10.5 mos). One D2-Gpnmb+ 8.5mo sample failed QC and was not included in the analysis. Quantile normalization was performed for all optic nerve head samples reported in the study. The complete dataset is linked below as a supplementary file.